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EVALUATION OF ANTITHROMBOCYTEPENIC POTENTIAL OF AMLA AND

KIWI FRUIT JUICES IN ANIMALS


6.1 Need of study:
Herbs and human health can never be separated. The vegetables and the fruits are herbs in a
way, they are essential for good health. However, there are many herbs, which have medicinal
values due to presence of certain enzymes, amino acids, alkaloids, vitamins etc 1.
Therapeutical properties of medical plants are very useful in healing various diseases and the
advantage of these medicinal plants is being 100% natural. Nowadays people are being
bombarded with thousand of unhealthy products, the level of sensibility in front of diseases is
very high and that's why the use of medicinal plants can represent the best solution2,3.
Platelets, or thrombocytes, are small, regularly shaped clear cell fragments (i.e. cells that do
not have a nucleus containing DNA), 23 m in diameter, which are derived from
fragmentation of precursor megakaryocytes. The average lifespan of a platelet is normally
just 5 to 9 days. Platelets are a natural source of growth factors. They circulate in the blood of
mammals and are involved in hemostasis, leading to the formation of blood clots. If the
number of platelets is too low, excessive bleeding can occur. However, if the number of
platelets is too high, blood clots can form (thrombosis), which may obstruct blood vessels and
result in such events as a stroke, myocardial infarction, pulmonary embolism or the blockage
of blood vessels to other parts of the body, such as the extremities of the arms or legs. An
abnormality or disease of the platelets is called a thrombocytopathy4, which could be either a
low number of platelets (thrombocytopenia), a decrease in function of platelets
(thrombasthenia), or an increase in the number of platelets (thrombocytosis).
Phyllanthus emblica (syn. Emblica officinalis), the Indian gooseberry, is a deciduous tree of
the Phyllanthaceae family. It is known for its edible fruit of the same name. Indian
gooseberry has undergone preliminary research, demonstrating in vitro antiviral and
antimicrobial properties5. There is preliminary evidence in vitro that its extracts induce
apoptosis and modify gene expression in osteoclasts involved in rheumatoid arthritis and
osteoporosis6. It may prove to have potential anti-cancer activity against some cancers 7. One
recent animal study found that treatment with E. ofcinalis reduced severity of acute
pancreatitis (induced by L-arginine in rats). It also promoted the spontaneous repair and
regeneration process of the pancreas occurring after an acute attack8.
The kiwifruit, often shortened to kiwi in many parts of the world, is the edible berry of a
cultivar group of the woody vine Actinidia deliciosa and hybrids between this and other

species in the genus Actinidia. The most common cultivars of kiwifruit are oval, about the
size of a large hen's egg (58 cm / 23 in long and 4.55.5 cm / 12 in diameter). It has a
fibrous, dull brown-green skin and bright green or golden flesh with rows of tiny, black,
edible seeds. The fruit has a soft texture and a unique flavour, and today is a commercial crop
in several countries, mainly in Italy, New Zealand, Brazil and Chile. Kiwifruit is often
reported to have mild laxative effects, due to its significant level of dietary fiber9.
As evident from folklore uses of amla and kiwi fruits, they cause alleviation from number of
diseases. Traditionally, one of the common remedial actions was for combating fall in platelet
count and their influence on blood disorders. Even though modern method of treatment is
successful in ameliorating the pains and discomforts of human being, there is still dearth of
discovery for effective management of thrombocytopenia. In line with this scenario, the
research envisaged is designed to to explore the role of amla fruit and kiwi fruit juices
separately as well as in combination on platelet count during collagen/heparin induced
thrombocytopenia in rats.
6.2 Review of literature:
There are disorders that reduce the number of platelets, such as heparin-induced
thrombocytopenia (HIT) or thrombotic thrombocytopenic purpura (TTP) that typically cause
thromboses, or clots, instead of bleeding. Platelets release a multitude of growth factors
including Platelet-derived growth factor (PDGF), a potent chemotactic agent, and TGF beta,
which stimulates the deposition of extracellular matrix. Both of these growth factors have
been shown to play a significant role in the repair and regeneration of connective tissues.
Other healing-associated growth factors produced by platelets include basic fibroblast growth
factor, insulin-like growth factor 1, platelet-derived epidermal growth factor, and vascular
endothelial growth factor. Local application of these factors in increased concentrations
through Platelet-rich plasma (PRP) has been used as an adjunct to wound healing for several
decades10-12.
Experimental preparations of leaves, bark or fruit have shown potential efficacy against
laboratory models of disease, such as for inflammation, cancer, age-related renal disease, and
diabetes13-15. A human pilot study demonstrated reduction of blood cholesterol levels in both
normal and hypercholesterolemic men16. Another very recent study with alloxan-induced
diabetic rats given an aqueous amla fruit extract has shown significant decrease of the blood

glucose as well as triglyceridemic levels and an improvement of the liver function caused by
a normalization of the liver-specific enzyme alanine transaminase (ALT) activity17. Although
fruits are reputed to contain high amounts of ascorbic acid (vitamin C), 445 mg/100g18, the
specific contents are disputed and the overall antioxidant strength of amla may derive instead
from its high density of tannins19. The fruit also contains other polyphenols: flavonoids,
kaempferol, ellagic acid and gallic acid19,20. Amla as it is known in India is also used to treat
hair disorders like premature falling and graying. It has been used to treat various diseases
through Ayurvedic medicine therapy dating back to many centuries.
In traditional Indian medicine, dried and fresh fruits of the plant are used. All parts of the
plant are used in various Ayurvedic/Unani Medicine [Jawarish Amla] herbal preparations,
including the fruit, seed, leaves, root, bark and flowers19. According to Ayurveda, amla fruit is
sour (amla) and astringent (kashaya) in taste (rasa), with sweet (madhura), bitter (tikta) and
pungent (katu) secondary tastes (anurasas) 19. Its qualities (gunas) are light (laghu) and dry
(ruksha), the post-digestive effect (vipaka) is sweet (madhura), and its energy (virya) is
cooling (shita) 18.
Raw kiwifruit is also rich in the protein-dissolving enzyme actinidin, (in the same family of
thiol proteases as papain), which is commercially useful as a meat tenderizer, but can be an
allergen for some individuals. Specifically, people allergic to latex, papayas or pineapples are
likely to also be allergic to kiwifruit. The fruit also contains calcium oxalate crystals in the
form of raphides. Reactions to these chemicals include sweating, tingling and sore mouth or
throat; swelling of the lips, tongue and face; rash; vomiting and abdominal pain, heartburn;
and, in the most severe cases, breathing difficulties, wheezing and collapse. The most
common symptoms are unpleasant itching and soreness of the mouth, with the most common
severe symptom being wheezing. Severe symptoms are most likely to occur in young
children. Actinidin also makes raw kiwifruit unsuitable for use in desserts containing milk or
any other dairy products which are not going to be served within hours, because the enzyme
soon begins to digest milk proteins. This applies to gelatin-based desserts as well, as the
actinidin will dissolve the collagen proteins in gelatin very quickly, either liquifying the
dessert, or preventing it from solidifying. However, the U.S. Department of Agriculture
suggests cooking the fruit for a few minutes before adding it to the gelatin to overcome this
effect22. Kiwifruit components, possibly involving vitamin E and omega-3 fatty acids from its
numerous edible seeds, have potential properties of a natural blood thinner. A study

performed at the University of Oslo in Norway reported consuming two to three kiwifruit
daily for 28 days significantly reduced platelet aggregation and blood triglyceride levels
(similar to popular mainstream aspirin therapy), potentially reducing the risk of blood clots22.
Kiwifruit is a natural source of carotenoids, such as provitamin A beta-carotene23, lutein and
zeaxanthin24.
One of the study evaluated the anti-hyperglycemic and lipid-lowering properties of Emblica
officinalis Gaertn. fruit in normal and diabetic human volunteers. The results indicated a
significant decrease (P < 0.05) in fasting and 2-h post-prandial blood glucose levels on the
21st day in both normal and diabetic subjects receiving 1, 2 or 3 g E. officinalis powder per
day as compared with their baseline values25.
As a part of ongoing research for novel natural cosmeceutical actives from plant extracts, a
study demonstrates that Phyllanthus emblica fruit extract has shown its efficacy in protection
against ultraviolet-B (UVB) irradiation-induced reactive oxygen species (ROS) and collagen
damage in normal human dermal fibroblasts. Emblica extract is a significantly better natural
active, with promising cosmeceutical benefits against photoaging26.
Emblica officinalis Gaertn. or Phyllanthus emblica Linn, commonly known as Indian
gooseberry or amla, is arguably the most important medicinal plant in the Indian traditional
system of medicine, the Ayurveda. Various parts of the plant are used to treat a range of
diseases, but the most important is the fruit. The fruit is used either alone or in combination
with other plants to treat many ailments such as common cold and fever; as a diuretic,
laxative, liver tonic, refrigerant, stomachic, restorative, alterative, antipyretic, antiinflammatory, hair tonic; to prevent peptic ulcer and dyspepsia, and as a digestive. Preclinical
studies have shown that amla possesses antipyretic, analgesic, antitussive, antiatherogenic,
adaptogenic, cardioprotective, gastroprotective, antianemia, antihypercholesterolemia, wound
healing,

antidiarrheal,

antiatherosclerotic,

hepatoprotective,

nephroprotective,

and

neuroprotective properties. In addition, experimental studies have shown that amla and some
of its phytochemicals such as gallic acid, ellagic acid, pyrogallol, some norsesquiterpenoids,
corilagin, geraniin, elaeocarpusin, and prodelphinidins B1 and B2 also possess antineoplastic
effects.

Amla

chemopreventive

is

also
effects,

reported
free

to

radical

possess

radiomodulatory,

scavenging,

antioxidant,

chemomodulatory,
anti-inflammatory,

antimutagenic and immunomodulatory activities, properties that are efficacious in the

treatment and prevention of cancer. This review for the first time summarizes the results
related to these properties and also emphasizes the aspects that warrant future research to
establish its activity and utility as a cancer preventive and therapeutic drug in humans27.
The fruit of Emblica officinalis has been used in the Ayurvedic system of medicine for the
treatment of different ailments and is also an ingredient of various traditional medicinal
herbal formulations in India and other countries. The protective effect of Emblica officinalis
fruit extract (EFE) against alcohol-induced brain mitochondrial dysfunction was investigated.
Administration of EFE to alcohol-treated rats, lowered the levels of NO, protein carbonyls,
and lipid peroxidation and elevated the activities of the antioxidant enzymes SDH, NADH
dehydrogenase, and cytochrome c oxidase and the content of cytochromes. The active
tannoid principles present in EFE with its antioxidant as well as NO scavenging properties
might have contributed to the observed protection against alcohol-induced brain
mitochondrial dysfunction28.
Emblica officinalis, an Indian medicinal plant, has marked antioxidant property. The effect of
seven days pretreatment of 300, 500 and 700 mg/kg doses of hydroalcoholic extract of E.
officinalis

(HAEEO)

administered

intraperitoneally

to

rats

was

evaluated

on

pentylenetetrazole (PTZ) induced seizures, cognitive deficit and oxidative stress markers viz
malondialdehyde (MDA) and glutathione. The 500 and 700 mg/kg ip doses of HAEEO
completely abolished the generalized tonic seizures and also improved the retention latency
in passive avoidance task. Further, HAEEO dose-dependently ameliorated the oxidative
stress induced by PTZ. These findings suggest the potential of HAEEO to be used as an
adjuvant to treatment with antiepileptic drugs29.
In order to gain insight into the in situ properties and localisation of kiwi pectin
methylesterase inhibitor (PMEI), a toolbox of monoclonal antibodies (MA) towards PMEI
was developed. Out of a panel of MA generated towards kiwi PMEI, three MA, i.e. MAKI9A8, MA-KI15C12 and MA-KI15G7, were selected. Thorough characterisation proved
that these MA bind specifically to kiwi PMEI and kiwi PMEI in complex with plant PME and
recognise a linear epitope on PMEI. Extract screening of green kiwi (Actinidia deliciosa) and
gold kiwi (Actinidia chinensis) confirmed the potential use of these MA as probes to screen
for PMEI in other sources. Tissue printing revealed the overall presence of PMEI in pericarp
and columella of ripe kiwi fruit. Further analysis on the cellular level showed PMEI label
concentrated in the middle lamella and in the cell-wall region near the plasmalemma.

Intercellular spaces, however, were either completely filled or lined with label. In conclusion,
the developed toolbox of antibodies towards PMEI can be used as probes to localise PMEI on
different levels, which can be of relevance for plant physiologists as well as food
technologists30.
6.3 Objective of study
The objective of the present research will be to explore the role of amla and kiwi fruit juices
on platelet count in animals with thrombocytopenia.
SPECIFIC OBJECTIVES:
LEVEL 1

To collect and authenticate the amla and kiwi fruit.

To standardize the method of preparation of amla and kiwi fruit.

To standardize the thrombocytopenic dose of collagen/heparin.

To established the effective dose of amla and kiwi fruit under normal conditions for
platelet count.

To ascertain the different concentrations of amla and kiwi fruit individually as well as
in combination for influencing the platelet count.

LEVEL 2

To study the extent of platelet enhancement by amla and kiwi fruit individually in
thrombocytopenic animals.

To know the platelet count when amla and kiwi fruit are used in combination during
thrombocytopenia.

LEVEL 3

To prepare suitable formulation of mixture containing effective concentrations of


amla and kiwi fruit juices.

To carryout stability study of the prepared formulation.

To periodically repeat the evaluation of formulation for platelet count.


MATERIALS AND METHODS:
7.1 Source of Data:

Data will be obtained from laboratory based studies by using Sprague dawley rats of either
sex weighing between 150-200 gms maintained at room temperature having free access to
food (std pellet diet), tap water ad libitum. These studies will be carried out in serum of
animal and the platelet count will be carried out in authenticated laboratories. Chemicals and
reagents will be procured from standard companies.
7.2 Method of Collection of Data:
Chemicals and reagents will be procured from standard companies. Collagen or Heparin
induced thrombocytopenia will be used as model to evaluate platelet changes. The data
collected will be based on animal experimentation as per the parameters studied under each
animal model.

Experimental protocol:
Multiple level studies will be carried out to ascertain the role of amla and kiwi fruit as platelet
enhancer.
Level 1
Animals will be divided into four groups (n=10)
Group I: collagen/heparin administration
Group II: Amla leaves juice administration
Group III: Kiwi juice administration
Group IV: Amla and Kiwi fruit juices in combination.
Blood samples will be collected before administration as shown above, as well as at 30
minutes, 1, 2, 4 and 8 hours after administration from all animals.
Level 2
Animals will be divided into three groups (n=10)
Group I: Amla juice + collagen/heparin administration
Group II: Kiwi juice + collagen/heparin administration

Group III: Amla and kiwi fruit juices in combination + collagen/heparin administration
Blood samples will be collected before administration as shown above, as well as at 30
minutes, 1, 2, 4 and 8 hours after administration from all animals.

Level 3
Animals will be divided into three groups (n=10)
Group I: prepared formulation of Amla and Kiwi fruit + collagen/heparin administration
Group II: vehicle used for dissolving collagen/heparin + collagen/heparin administration
Group III: Excipients of the formulation + collagen/heparin administration
Blood samples will be collected before administration as shown above, as well as at 30
minutes, 1, 2, 4 and 8 hours after administration from all animals.
Stability studies of the prepared formulation will be carried out and periodically platelet
count will be checked in animals treated with formulation.

Induction of thrombocytopenia31,32
Intravenous administration of collagen, PAF (platelet activating factor) or thrombin activates
thrombocytes leading to a maximal thrombocytopenia within a few minutes. The effect is
reinforced by additional injections of epinephrine. Activation of platelets leads to intravasal
aggregation and temporary sequestration of aggregates in the lungs and other organs.
Depending on the dose of agonist, this experimentally induced reduction of the number of
circulating platelets is reversible within 60 min after induction.

PROCEDURE
Male Sprague dawley rats of either sex weighing between 150-200 gms will be used. Animals
receive the test compound or the vehicle (controls) by oral, intraperitoneal or intravenous
administration. After the end of the absorption time (p.o. 60 min, i.p. 30 min, i.v. variable),

the thrombocytopenia-inducing substances collagen are injected slowly33. Blood will be


collected from tail Approx. 50100 l blood are collected into potassium- EDTA-coated tubes
at times 5, 10 and 15 min following the injection of the inducer. The number of platelets and
leukocytes is determined within 1 h after withdrawal in 10 l samples of whole blood using a
microcellcounter. The percentage of thrombocytes (or leukocytes) is determined in vehicle
control and dosage groups at the different times following injection of the inducer relative to
the initial value of control or dosage group, respectively. Calculated percent values of
controls are taken as 100%. Percent inhibition of thrombocytopenia (or leucocytopenia) is
calculated in dosage groups relative to controls.
Statistical analysis
Statistical significance is evaluated by means of the unpaired Students t-test when
comparisons were made between two groups. Further group analysis will be done by using
one-way analysis of variance (ANOVA) followed by Dunnet comparison test. The values will
be expressed as mean SEM and p < 0.05 will be considered significant.
7.3 Does the study require any investigation or interventions to be conducted on patients
or the human or animals? If so please describe briefly:
YES
Study requires investigation on animals. The effects of the drug will be studied on various
parameters using rats as experimental animals.
7.4 Has ethical clearance been obtained from your institute ?
Ethical Committee approval letter is enclosed.

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